The objective of this proposal is to pursue studies on the regulation of intestinal smooth muscle function by peptide and non-peptide transmitters of the enteric nervous system. Our recent studies have identified receptors for CCK, tachykinins, bombesin, 5-HT and adenosine in intestinal muscle coupled to distinct, interactive signaling pathways. The same receptor (e.g., CCK-A) was coupled via different G proteins to PI hydrolysis by PLC-beta1, and to PC hydrolysis by PLA2, PC-PLA2, PC-PLC and PLD. The messengers derived from these phospholipids (IP3, DAG and arachidonic acid) were shown to elicit cell-specific responses. The studies provided the first evidence of IP3-dependent Ca2+ release in intestinal circular muscle and cADP ribose-modulated Ca2+ release via ryanodine-sensitive Ca2+ channels in longitudinal muscle; Ca2+ release in longitudinal muscle was triggered by arachidonic acid-mediated Ca2+ channels in longitudinal muscle; Ca2+ release in longitudinal muscle was triggered by arachidonic acid-mediated Ca2+ influx. Initial contraction was shown to be mediated by MLC kinase and sustained contraction by a Ca2+- dependent or -independent PKC isoform, depending on the agonist. Accordingly, the first specific aim of the current proposal is to characterize the receptor subtypes for opioid peptides (mu, sigma kappa), neuropeptide Y (Y1 and Y2), somatostatin (SSTR-2 and SSTR-3) and acetylcholine (M2 and m3) and the signaling pathways to which they are coupled. Preliminary studies show that these receptors are coupled to distinct G proteins (Gi1, Gi2, G13, Go) and PLC-beta isoforms. The receptor subtypes will be identified by selective receptor protection and the steps in the signaling pathways by specific G protein and effector enzyme antibodies. The second specific aim is to characterize the novel, cell-specific Ca2+ -mobilizing messengers, cADP ribose, cGMP and arachidonic acid. Preliminary studies show agonist-stimulated synthesis of cADP ribose from beta-NAD+ in longitudinal muscle. A selective bioassay based on Ca2+ release in longitudinal muscle cells and using the specific inhibitor, 8-amino-cADP ribose, has been developed to measure endogenous cADP ribose. cGMP was shown to induce Ca2+ release via IP3- and cADP riboseinsensitive Ca2+ channels. The third specific aim is to characterize the role of MLC kinase in initial contraction and Ca2+ -dependent and - independent PKC isoforms in sustained contraction. The role of two-actin associated regulatory proteins, calponin and caldesmon, in sustained contraction induced by G protein-coupled agonists, bombesin (a hybrid agonist/growth factor), and phorbol ester, will be determined with emphasis on phosphorylation of these proteins directly by PKC or PKM and indirectly by PKC-dependent MAP kinase. The studies encompassed by these specific aims should advance our understanding of the regulation of intestinal muscle contraction.